The elucidation of the human and other genomes (1,2) has spurred efforts to identify proteins expressed by cells in various tissues, i.e. the proteome. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) is one of the major tools being employed for the analysis of peptides generated by enzymatic digestion of expressed proteins. Trypsin is the most commonly used enzyme because the predictability of its cleavages facilitates identification of proteins by database searching. A difficulty encountered with this strategy is that there is a great variability in the signal intensity for peptide depending on their composition. In many cases peptides which are known to be generated are not detected. It has been observed that the detection of arginine-containing peptides is much more sensitive than lysine-containing peptides (3). Conversion of lysine residues in peptides to homo-arginine by guanidinylation has been shown to increase the sensitivity of detection of these peptides (4,5,6). The reason for this increased sensitivity is not fully understood and the use of guanidinylation for increasing the sensitivity of MALDI peptide detection is based only on an empirical observation. A severe limitation of this approach is that it is applicable only to peptides containing lysine. In enzymatic digests, other than trypsin, the majority of the peptides are not lysine peptides and this methodology is not applicable.
In vacuo reaction of amino groups with iodomethane in proteins or peptides produces a trimethylated quaternary ammonium derivative with a permanent positive charge (7,8,910). There is no known theory that predicts that this derivatization would increase the sensitivity of detection of peptides by MALDI MS. However, this possibility has never been tested primarily because iodomethane has not been used for chemical modification of peptides or proteins in aqueous solution due to its low solubility in water. Currently, modification procedures are carried out under aqueous conditions that require several manipulative procedures. In vacuo trimethylation requires only one step and is easier to carry out than modification procedures under aqueous conditions. In addition it has several other significant technical advantages:    1) It requires no solvent removal, clean-up steps or any other manipulation prior to preparing the sample for mass spectrometric analysis.    2) Methylation can be carried out on much smaller amounts of peptide or protein than a solvent-based modification procedure such as guanidinylation.    3) Very small amounts of reagent are required which permits the cost effective use of isotopically enriched (13C, 14C, CD3 CT3) reagent for special applications.    4) Guanidinylation can only be carried out on ε-amino groups whereas methylation can be carried out on both α-amino and ε-amino groups.    5) The in vacuo methylation can be used to differentiate between peptide and non-peptide material greatly simplifying the interpretation of the mass spectra. There is therefore a need for a method of detecting a peptide using mass spectrometry that involves in vacuo trimethylation of the peptide.